Atherosclerosis results in part from the accumulation of lipoproteins in the arterial wall, recruitment of monocytes/macrophages to the region, and formation of lipid-laden macrophages, known as foam cells. Foam cell formation is dependent on interactions with lipoproteins and subsequent cellular cholesterol transport among specific intracellular compartments. This project will examine how characteristics of both the lipoprotein and the macrophage lead to foam cell formation and the pathogenesis of atherosclerosis. Several key aspects of this process will be examined. First, many of the lipoproteins in the wall of blood vessels become aggregated and tightly bound to extracellular matrix components, and this prevents their internalization by simple endocytic processes. When macrophages encounter these aggregated lipoproteins, there is a prolonged period of contact before the lipoprotein is internalized. Preliminary data indicate that macrophages form an unusual extracellular, acidic, digestive compartment during this contact period. The acidification of this extracellular compartment will be characterized further using quantitative fluorescence microscopy, and the mechanism for acidification will be examined. The structure of the extracellular compartment will be examined using intermediate voltage electron microscopy and tomographic imaging. The mechanism for delivering acid hydrolases into the compartment will also be examined, including the role of calcium signaling and specific SNARE molecules in mediating lysosomal fusion with the plasma membrane. The presence of lysosomal acid lipase in this compartment would lead to hydrolysis of cholesterol esters and release of free sterol, which can be inserted locally in to the plasma membrane. Well- characterized fluorescent sterols and their ester derivatives will be incorporated into lipoproteins to monitor this process directly. Other catabolic processes (e.g., protein degradation) in this compartment will be investigated. Delivery of cholesterol to the plasma membrane leads to activation of signal transduction mechanisms, including the small GTPase, Rac, leading to increased actin polymerization. The consequences of sterol delivery from aggregated lipoproteins to macrophages on Rac, Rho and PI3-kinase activity and actin assembly will be examined. The possibility that increased actin assembly enlarges the extracellular, acidic, digestive compartment will be examined using inhibitors of these signaling proteins. Different classes of macrophages may respond differently to aggregated lipoproteins. The interactions of macrophages derived from classical and nonclassical monocytes with aggregated lipoproteins will be compared in tissue culture. The abundance of macrophages and foam cells derived from classical and nonclassical Mo subsets in atherosclerotic lesions in mouse models of atherosclerosis will also be examined. PUBLIC HEALTH RELEVANCE: This project examines the early steps leading to an atherosclerotic lesion as a consequence of the interaction between an immune system cell, the macrophage, with lipoproteins in the wall of blood vessels. These atherosclerotic lesions can lead to heart disease and stroke, which are major causes of disability and death of Americans. Research findings based on these studies may lead to new treatments to prevent or reverse the formation of atherosclerotic lesions.